Identifying the etiological agent of sepsis or community-acquired pneumonia (CAP) greatly improves the clinician's ability to manage the patient. Even with all of the newly developed technologies of molecular diagnostics, there have been essentially no recent innovations in diagnostic testing for these significant and often life threatening diseases. Blood culture, microbial sugar utilization-based identification, and empirical chemotherapy have dominated diagnostics and patient management decisions for several years. Empirical treatment - often inappropriate - is partially responsible for the epidemic spread of multidrug resistant pathogens. Bacterial, fungal, and viral agents can all be concurrently detected and identified with nucleic acid amplification and hybridization methods. Innovative approaches to nucleic acid isolation from clinical samples, coupled with multiplex PCR amplification, and detection on electronic microarrays has the potential to significantly advance the ability of the clinician to correctly identify the underlying pathogen and manage the disease. This proposal describes a program to merge innovative, yet proven technology for sample preparation, high multiplex amplification, and detection and differentiation with biomarkers identifying at least 17 agents of CAP and sepsis with an automated system for the clinical microbiology laboratory. The investigators have all made significant contributions in commercializing new molecular diagnostic systems for detection and identification of pathogens in the areas of: respiratory viruses, blood banking, sexually transmitted disease, bioterrorism, CAP, and sepsis. The specific aims are: (1) Optimize a multiplex PCR system including bacterial and viral CAP and sepsis agent targets;(2) Select a suitable sample preparation chemistry;(3) Optimize the assay on electronic microarrays;(4) Develop self-contained modules for the three assay steps;(5) Integrate the modules into a prototype disposable, on a prototype instrument;(6) Develop processes and specifications for critical raw materials;and (7) Test the prototype integrated assay in a clinical study. At the completion of the research program, a number of useful tools will be available to improve molecular diagnostic methods for CAP and sepsis (and potentially other diseases), and a fully integrated automated assay that will be in the final stages of commercialization.